The long range objective of this research is to understand the mechanism(s) of insulin resistance in muscle of obese and diabetic patients. There is considerable evidence that muscle glucose transport is decreased in obesity and non-insulin dependent diabetes mellitus (NIDDM). We have observed a 50% depression in the maximal rate of glucose transport but only a 20% decrease in the number of glucose transporters in insulin resistant muscle, suggesting that there are additional defects in either insulin signalling or in translocation of glucose transporter protein to the cell membrane. We propose mechanistic studies to investigate the cause(s) of insulin resistance in muscle. Through a cooperative research effort with the Department of Surgery we have a unique opportunity to obtain muscle tissue from a substantial number of morbidly obese patients with diabetes (NIDDM) and we plan to use this tissue to investigate the cause of insulin resistance. Abdominal muscle will be obtained from morbidly obese patients undergoing gastric bypass surgery and from nonobese patients undergoing elective abdominal surgery. From the muscle biopsies thin muscle fiber strips are mounted in clamps at resting length and incubated in vitro. The mechanisms causing insulin resistance in human muscle will be investigated using this unique muscle preparation. The hypothesis for these studies is that hypoxia (or muscle contraction) and phosphatase inhibitors overcome insulin resistance by causing the accumulation of one or more phosphorylated proteins essential for signalling the translocation of glucose transporters to the cell membrane. The following specific aims address this hypothesis: (1) To determine whether stimulation of glucose transport by hypoxia is normal in insulin resistant muscle of obese human patients and whether hypoxia will enhance insulin responsiveness in these muscles as seen in obese animal models; (2) To investigate whether the insulin stimulated phosphorylation of proteins is altered in insulin resistant muscle of obese patients. In addition, we will determine the phosphorylation profile of proteins in response to hypoxia, hypoxia plus insulin, and phosphatase inhibitors to ascertain whether there are common intermediates in the stimulation by these pathways; and (3) To study glucose transporter translocation in response to insulin, hypoxia and phosphatase inhibitors in muscle of lean and obese individuals.